Biomedical devices with antimicrobial coatings

ABSTRACT

Biomedical devices with antimicrobial coatings are provided. One or more surfaces of the device are coated with a cationic peptide, cationic proteins, or mixtures thereof to impart antimicrobial properties to the surface.

RELATED APPLICATIONS

This patent application is a divisional of U.S. Ser. No. 10/325,255,filed on Dec. 19, 2002, now U.S. Pat. No. 7,282,214.

FIELD OF THE INVENTION

This invention relates to coated devices. In particular, the inventionprovides biomedical devices on the surfaces of which antimicrobialcoatings of a synthetic cationic peptide are formed.

BACKGROUND OF THE INVENTION

Devices for use in and on the human body are well known. The chemicalcomposition of the surfaces of such devices plays a pivotal role indictating the overall efficacy of the devices. Additionally, it is knownthat providing such devices with an antimicrobial surface isadvantageous.

A wide variety of bactericidal and bacteriostatic coatings have beendeveloped. For example, cationic antibiotics, such as polymyxin,vancomycin, and tetracycline have been used as coatings for contactlenses. Further, metal chelating agents, substituted and unsubstitutedpolyhydric phenols, aminophenols, alcohols, acid and amine derivatives,and quartemary ammonium have been used as antimicrobial agents forcontact lenses. U.S. Pat. No. 5,472,703 discloses certain lipidcompounds as antimicrobial agents for contact lenses.

However, the use of these known antimicrobial coatings hasdisadvantages. With the use of antibiotic coatings, microorganismsresistant to the antibiotics may develop. Chelating agent use fails toaddress the numbers of bacteria that adhere to the device. Some of theprior art coatings, for example phenol derivatives and cresols, canproduce ocular toxicity or allergic reactions. Quarternary ammoniumcompounds are problematic because of their irritancy. Thus, a needexists for safe and effective antimicrobial coatings that overcomes atleast some of these disadvantages.

U.S. Ser. No. 09/516,636 discloses that protamine, melittin, cecropin A,nisin, or combinations thereof, may be used as surface coatings toreduce adherence of bacteria to a device's surface and/or reduce growthof bacteria adhered to a device. Unfortunately those peptides have beenfound to be toxic in certain concentrations or have a limited spectrumof antimicrobial activity.

Subbalakshmi et al., FEBS Letters, 448, pgs. 62-66 (1999) discloses thatthe C-terminal 15 amino acid residues of melittin, retain theirantibacterial activity but has greatly reduced haemolytic activity.Juvvadi et al. disclose placing the C-terminal of melittin at theN-terminal of synthetic peptides reduced mammalian cell cytotoxicity(Juvvadi et al., J. Am. Chem. Soc., vol. 118, pgs. 8989-8997 (1996)).However, the range of bacteria that were inhibited by the C-terminalpeptide were decreased and the amount of peptide needed to inhibit thosebacteria was increased (Subbalakshmi et al., FEBS Letters, 448, pgs.62-66 (1999)).

Mixtures of cationic peptides have also been disclosed. For example,synthetic peptides containing lysine as the cationic moiety have beensynthesized (Mor et al., J. Biol. Chem., vol. 269, pgs. 31635-31641(1994)) as has a mixture of protamine and melittin (Aliwarga et al.,Clim. Exp. Opthalmol., vol. 29, pgs. 157-160 (2001)).

Synthetic peptides that incorporate the active moieties from differentcationic peptides in one single molecule have also been synthesized. Forexample, a series of peptides made from combinations of cecropin A andmelittin were disclosed to retain most of their antibacterial efficacy(Boman et al., FEBS Letters, vol. 259, pgs. 103-106 (1989)). A hybrid ofcecropin A and melittin was also synthesized and disclosed to reducesigns of infection and inflammation in an experimental model ofmicrobial keratitis (Nos-Barbera et al., Cornea, vol. 259, pgs, 101-106(1996)). However, the toxic regions of melittin were also retained andwould be expected to induce toxicity in mammalian cells.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the % lysis sheep red blood cells relative towater for various peptides.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention provides biomedical devices with an antimicrobialcoating and processes for the production of the biomedical devices. Itis an unexpected discovery of the invention that certain syntheticpeptides may be used to provide antimicrobial coatings for biomedicaldevices. Specifically, the synthetic antimicrobial peptides of thepresent invention comprise the 15-26 segment of mellitin, segment A:

TLISWIKNKRKQand the 1-17 segment of protamine, segment B:

RPRVSRRRRRRGGRRRRpresent anywhere in a peptide. The peptides may further comprise a thirdsegment C, which may be any linking group which does not inhibit theantimicrobial activity of the peptide or induce toxicity in mammaliancells, and which includes spacers of 0 to about 10 amino acids. Aminoacids, as defined herein, refer to any structure with the chemicalformula —HN—(CR¹R²)_(n)—CO— wherein n is an integer between 1 and 21, R¹and R² are independently selected from the group consisting of H,straight or branched alkyl groups having 1 to 4 carbon atoms, straightor branched hydroxy groups having 1-2 carbon atoms, straight or branchedalkylthio groups having 1 to 3 carbon atoms, carbamoyl groups having 1to 3 carbon atoms, carboxy groups having 1 to 3 carbon atoms, primaryand secondary amino groups having 1 to 4 carbon atoms and 1 to 3nitrogen atoms, benzyl, phenol, phenyl indoles and N,N-pyrroles.Preferably n is an integer between 1 and 10 and at least one of R¹ andR² is H and the other is selected from the above. The A, B and Csegments of the antimicrobial synthetic peptide may be in any order andmay be repeated in part or whole. In a preferred embodiment, the A and Bsegments are in terminal positions and in another preferred embodimentthe synthetic antimicrobial peptide has the formula ACB or BCA and Ccomprises up to 5 amino acids.

The invention also includes synthetic antimicrobial peptides that areconservative variations of those peptides exemplified herein. The term“conservative variation” as used herein denotes a polypeptide in whichat least one amino acid is replaced by another, biologically similarresidue. Examples of convservative variations include the substitutionof one hydrophobic residue, such as isoleucine, valine, leucine,alanine, cysteine, glycine, phenylalanine, proline, tryptophan,tyrosine, norleucine or methionine for another, or the substitutions ofone polar residue for another such as the substitution of arginine forlysine, glutamic acid for aspartic acid or glutamine for asparagine andthe like. Neutral hydrophilic amino acids that can be substituted forone another include asparagine, glutamine, serine and threonine.

In particular, it is one discovery of the invention that melimine,protattin and mixtures thereof when used as surface coatings, reducesadherence of bacteria to a device's surface, reduces growth of bacteriaadhered to a device, or both by greater than about 40 percent.

In one embodiment, the invention provides a biomedical devicecomprising, consisting essentially of, and consisting of at least onesurface comprising, consisting essentially of, and consisting of acoating effective amount of at least one synthetic antimicrobialpeptide. In yet another embodiment, a method for manufacturingbiomedical devices comprising, consisting essentially of, and consistingof contacting at least one surface of a biomedical device with a coatingeffective amount of at least one synthetic antimicrobial peptide isprovided. In still another embodiment of the invention, a second methodfor the manufacturing of biomedical devices comprising, consistingessentially of, and consisting of contacting at least one surface of abiomedical device with a coating effective amount of at least onesynthetic antimicrobial peptide is provided. In yet another embodimentthe present invention provides a method for manufacturing a biomedicaldevice comprising, consisting essentially of, and consisting of addingat least one polymerizable synthetic antimicrobial peptide to a reactivemixture and polymerizing said reactive mixture to form a biomedicaldevice.

By “biomedical device” is meant any device designed to be used while inor on either or both human tissue or fluid. Examples of these devicesinclude but are not limited to catheters, implants (including, but notlimited to heart valves), stents, fluid collection bags, sensors,hydrogel bandages, tubing, carriers for antibiotic, diagnostic andtherapeutic agents, and ophthalmic devices. In some embodimentscatheters are a preferred medical device. A class of preferredbiomedical devices includes ophthalmic devices, particularly contactlenses.

As used herein, the terms “lens” and “opthalmic device” refer to devicesthat reside in or on the eye. These devices can provide opticalcorrection, wound care, drug delivery, diagnostic functionality or maybe cosmetic. The term lens includes but is not limited to soft contactlenses, hard contact lenses, intraocular lenses, overlay lenses, ocularinserts, and optical inserts.

In a preferred embodiment, the biomedical device is an ophthalmic lensincluding, without limitation, contact or intraocular lenses. Morepreferably, the device is a contact lens, most preferably a soft contactlens.

The term “peptide” or “polypeptide”, as used herein, refers to the basicchemical structure of polypeptides that is well known and has beendescribed in textbooks and other publications in the art. In thiscontext, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types. It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, can be modified in a given polypeptide, eitherby natural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques which are wellknown to the art. Even the common modifications that occur naturally inpolypeptides are too numerous to list exhaustively here, but they arewell described in basic texts and in more detailed monographs, as wellas in a voluminous research literature, and they are well known to thoseof skill in the art. Among the known modifications which can be presentin polypeptides of the present are, to name an illustrative few,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. Such modifications are well known to those of skill andhave been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as, for instance PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York(1993). Many detailed reviews are available on this subject, such as,for example, those provided by Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., (1990), Meth. Enzymol.182, 626-646 and Rattan et al., “Protein Synthesis: PosttranslationalModifications and Aging”, (1992) Ann. N.Y. Acad. Sci. 663, 48-62. Itwill be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptides(both linear and non-linear) can be generated as a result ofposttranslational events, including natural processing event and eventsbrought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides can be synthesizedby non-translation natural process and by entirely synthetic methods, aswell. Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. In fact, blockage of the amino or carboxyl group in apolypeptide, or both, by a covalent modification, is common in naturallyoccurring and synthetic polypeptides and such modifications can bepresent in polypeptides of the present invention, as well. For instance,the amino terminal residue of polypeptides made in E. coli or othercells, prior to proteolytic processing, almost invariably will beN-formylmethionine. During post-translational modification of thepeptide, a methionine residue at the NH₂-terminus can be deleted.Accordingly, this invention contemplates the use of both themethionine-containing and the methionineless amino terminal variants ofthe protein of the invention. The modifications that occur in apolypeptide often will be a function of how it is made. For polypeptidesmade by expressing a cloned gene in a host, for instance, the nature andextent of the modifications in large part will be determined by the hostcell posttranslational modification capacity and the modificationsignals present in the polypeptide amino acid sequence. For instance, asis well known, glycosylation often does not occur in bacterial hostssuch as, for example, E. coli. Accordingly, when glycosylation isdesired, a polypeptide should be expressed in a glycosylating host,generally a eukaryotic cell. Insect cells often carry out the sameposttranslational glycosylations as mammalian cells and, for thisreason, insect cell expression systems have been developed to expressefficiently mammalian proteins having native patterns of glycosylation,inter alia. Similar considerations apply to other modifications. It willbe appreciated that the same type of modification can be present in thesame or varying degree at several sites in a given polypeptide. Also, agiven polypeptide can contain many types of modifications. In general,as used herein, the term polypeptide encompasses all such modifications,particularly those that are present in polypeptides synthesizedrecombinantly by expressing a polynucleotide in a host cell.

As used herein “Melimine” refers to a polypeptide comprising an aminoacid sequence of:

T-L-I-S-W-I-K-N-K-R-K-Q-R-P-R-V-S-R-R-R-R-R-R-G-G- R-R-R-RWherein: T is threonine, L is leucine, I is isoleucine, S is serine, Wtryptophan, K is lysine, N is asparagine, R is arginine, Q is glutamine,P is Proline, V is valine. As used herein, L-melimine comprises theabove amino acid sequence as it exists naturally. Optical isomers ofamino acids undergo spontaneous nonenzymatic racemisation. This ratevaries for each amino acid at a given temperature or pH (storageconditions), but is more rapid in D than L isomers. As used herein L- orD-peptides will comprise about 99% L or D isomers, respectively at a pHof 7 and a temperature of about 25° C.

L-amino acids are the naturally occurring form in biological systems,therefore D-isomers are more resistant to enzymatic breakdown and mayhave an increased persistance. This property may be exploited by the useof mixtures of the stereoisomers to give desired levels of activity andlongevity for a particular application. So, for applications where longterm persistence is desired, the use of a stereoisomeric mixture havinga predominance (greater than about 50% and preferably greater than about70%) D isomer may be preferred. Where greater antibacterial activity isdesired the use of a stereoisomeric mixture having a predominance(greater than about 50% and preferably greater than about 70%) L isomermay be preferred.

As used herein “Protattin” refers to a polypeptide comprising an aminoacid sequence of

R-P-R-V-S-R-R-R-R-R-R-G-G-R-R-R-R-T-L-I-S-W-I-K-N- K-R-K-Q.Wherein the amino acids are as defined above.

For purposes of the invention, generally the cationic peptide used issubstantially purified.

As used herein, the term “substantially purified” means that the proteinor biologically active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the protein is derived, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus,protein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of heterologous protein (also referred to herein as a“contaminating protein”). When the protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, or 5% of the volume of the proteinpreparation. When the protein is produced by chemical synthesis, it ispreferably substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. Accordingly suchpreparations of the protein have less than about 30%, 20%, 10%, 5% (bydry weight) of chemical precursors or compounds other than thepolypeptide of interest.

Synthetic antimicrobial peptides useful in the invention can besynthesized chemically using standard peptide synthesis techniques.Alternatively, synthetic antimicrobial peptides useful in the presentinvention can be synthesized in an in vitro translation and/ortranscription system.

Synthetic antimicrobial peptides may be synthesized using conventionalsolid-phase peptide and solution peptide synthesis protocols. Suchmethods are well known to those skilled in the art. What follows is adescriptive way of making a synthetic antimicrobial petide using thesolid phase synthetic technique, but in no way limits the scope of thisinvention to this method. The synthesis may be performed on any suitablesynthetic resin. Suitable resins include insoluble cross-linkedpolystyrene resin and the like. The amino acids are generally protectedusing fluorenylmethoxycarbonyl groups and the like and activated withN-hydroxybenzotriazole and, but not necessarily, diisopropylcarbodiimide(DIC) to facilitate their coupling. The completed peptide is cleavedfrom the resin using, but not limited to trifluoroacetic acid orammonia, and the resulting material purified by reverse-phasehigh-performance liquid chromatography (HPLC) and/or dialysis, afterwhich candidate material is freeze-dried from a water/acetonitrilemixture to a dry powder.

Synthetic antimicrobial peptides useful in the invention can also beproduced using an in vitro translation and/or transcription system. Suchmethods are known to those skilled in the art. For example, syntheticmRNA encoding a Melimine or Protattin can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts. Alternatively, synthetic DNAcomprising the coding sequence for a Melimine or Protattin under thecontrol of a T7 promoter can be efficiently transcribed and translated,in an in vitro transcription and translation system, such as the TNT T7coupled Reticulocyte Lysate System, which is commercially available fromPromega. The resulting polypeptide can be purified by method describedherein.

L-melimine, protattin, or combinations thereof may be adsorbed topolymer surfaces of a biomedical device. The synthetic antimicrobialpeptides may be used on any surface, but most advantageously are usedwith negatively charged surfaces.

Alternatively, the synthetic antimicrobial peptides may befunctionalized with acryloyl or methacryloyl groups) that can be addedinto the monomer mix which is then reacted to form a biomedical devicehaving synthetic antimicrobial peptide in the bulk of the polymerforming the device, as well as on the device's surface.

The synthetic antimicrobial peptides alternatively may be bound to thepolymer surfaces. This may be either a direct reaction or, preferably, areaction in which a coupling agent is used. For example, a directreaction may be accomplished by the use of a reagent of reaction thatactivates a group in the surface polymer or the synthetic antimicrobialpeptide making it reactive with a functional group on the peptide orpolymer, respectively, without the incorporation of a coupling agent.For example, one or more amine or alcohol or thiol groups on thesynthetic antimicrobial peptide may be reacted directly withisothiocyanate, acyl azide, N-hydroxysuccinimide ester,pentafluorophenoxy ester, sulfonyl chloride, an aldehyde, glyoxalepoxide, carbonate, aryl halide, imido ester, tosylate ester or ananhydride group on the polymer.

In an alternative embodiment, coupling agents may be used. Couplingagents useful for coupling the cationic peptide or protein to thedevice's surface include, without limitation, N,N′-carbonyldiimidazole,carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(“EDC”), dicyclohexyl carbodiimide,1-cylcohexyl-3-(2-morpholinoethyl)carbodiimide, diisopropylcarbodiimide, or mixtures thereof. The carbodiimides also may be usedwith N-hydroxysuccinimide or N-hydroxysulfosuccinimide to form estersthat can react with amines to form amides.

Amino groups also may be coupled to the polymer by the formation ofSchiff bases that can be reduced with agents such as sodiumcyanoborohydride and the like to form hydrolytically stable amine links.Coupling agents useful for this purpose include, without limitation,N-hydroxysuccinimide esters, such as dithiobis(succinimidylpropionate),3,3′-dithiobis(sulfosuccinimidylpropionate), disuccinimidyl suberate,bis(sulfosuccinimidyl) suberate, disuccinimidyl tartarate and the like,imidoesters, including, without limitation, dimethyl adipimate,difluorobenzene derivatives, including without limitation1,5-difluoro-2,4-dinitrobenzene, bromofunctional aldehydes, includingwithout limitation gluteraldehyde, and bis epoxides, including withoutlimitation 1,4-butanediol diglycidyl ether. One ordinarily skilled inthe art will recognize that any number of other coupling agents may beused depending on the functional groups present on the device's surface.

One ordinarily skilled in the art also will recognize that, if thedevice's surface does not contain suitable reactive groups, suchsuitable groups may be incorporated into the polymer by any conventionalorganic synthesis methods. Alternatively, the reactive groups may beintroduced by the addition of polymerizable monomers containing reactivegroups into the monomer mixture used to form the polymer.

Examples of polymer surfaces onto which the synthetic antimicrobialpeptides may be adsorbed or bonded are surfaces formed from, withoutlimitation, polymers and copolymers of styrene and substituted styrenes,ethylene, propylene, acrylates and methacrylates, N-vinyl lactams,acrylamides and methacrylamides, acrylonitrile, acrylic and methacrylicacids as well as polyurethanes, polyesters, polydimethylsiloxanes andmixtures thereof. Such polymers may include hydrogels and siliconehydrogels. Preferably, lightly crosslinked polymers and copolymers of2-hydroxyethylmethacrylate (“HEMA”) are used. By “lightly crosslinked”is meant that the polymer has a low enough crosslink density so that itis soft and elastic at room temperature. Typically, a lightlycrosslinked polymer will have about 0.1 to about 1 crosslinking moleculeper about 100 repeating monomer units. Examples of suitable lightlycrosslinked HEMA polymers and copolymers include without limitation,etafilcon A and copolymers of glycerol methacrylate and HEMA. Alsopreferably, silicone hydrogels, especially those of hydrophilicmonomers, such as N,N-dimethylacrylamide, are used.

In one embodiment of the process for making the device of the invention,the surface to be coated is contacted with the L-melimine, protattin orcombinations thereof in any convenient manner. Preferably, the coatingcomprises L-melimine. For example, the device may be placed in asolution of L-melimine and solvent into which the medical device isplaced. As an alternative, the device's surface may first be treatedwith a coupling agent and the surface then placed in a solution of theselected synthetic antimicrobial peptide. As yet another alternative thesynthetic antimicrobial peptide may be reacted alone with the polymersurface.

In certain embodiments the free NH₂ groups of the syntheticantimicrobial peptides of the present invention are attached to apolymeric surface containing reactive COOH groups.

Suitable solvents for use in the invention are those that are capable ofdissolving the selected synthetic antimicrobial peptide, such asL-melimine, protattin singly or in combination. Preferably, the coatingprocess is carried out in water, alcohol, or mixtures thereof. EDC iseffective in aqueous solutions and, thus, is a preferred coupling agent.

The coupling agents may be used alone or in combination with agentscapable of stabilizing any reactive intermediate formed. For example,EDC may be used with N-hydroxysuccinimide as a stabilizer. Additionally,it may be desirable to adjust the pH. Preferably, the pH is adjusted toabout 6.5 to about 8.0, more preferably about 7.0 to about 7.5.

Alternatively, latent reactive components may be added to the monomermix where the selected polymer does not have suitable carboxylic acidfunctionality. Suitable latent reactive components include, withoutlimitation, ester compounds of the formula R—CO—L wherein R comprises agroup capable of cationic, anionic or free radical polymerization and Lis a leaving group. Suitable R groups include monovalent groups that canundergo free radical and/or cationic polymerization comprising up to 20carbon atoms. Preferred R groups comprise free radical reactive groups,such as acrylates, styryls, vinyls, vinyl ethers, C₁₋₆alkylacrylates,acrylamides, C₁₋₆alkylacrylamides, N-vinyllactams, N-vinylamides,C₂-C₁₂alkenyls, C₂₋₁₂alkenylphenyls, C₂₋₁₂alkenylnaphthyls, orC₂₋₆alkenylphenylC₁₋₆alkyls or a cationic reactive group such as vinylethers or epoxide groups and mixtures thereof. Particularly preferred Rgroups include methacrylates, acryloxys, methacrylamides, acrylamides,and mixtures thereof.

Suitable L groups are stable under reaction conditions, and protect thecarboxylate group and leave readily under coating conditions. Suitable Lgroups include alkyl esters, phenyl esters, hydroxy para-nitroaryls,p-nitrophenyl esters, N-hydroxylamine derivatives, and tosylate estersall of which may be substituted or unsubstituted. Preferred L groupsinclude t-butyl esters, 2,4,5-trichlorophenyl esters, pentafluorophenylesters, N-hydroxysuccinimide esters, N-hydroxy-oxo-dihydrobenzotriazinederivatives, 1-hydroxybenzotriazole esters and combinations thereof.Preferred suitable L groups include pentafluorophenyl esters, tosylateesters, and N-hydroxysuccinimide esters, and mixtures thereof. Preferredlatent reactive compounds include pentafluoromethacrylate andN-acryloxysuccinimide and mixtures thereof and the like.

A coupling effective amount of the selected coupling agent or reactivecomponent is used which amount is an amount sufficient to couple thesynthetic antimicrobial peptide to the device surface. The preciseamount of coupling agent used will depend on the surface's chemistry aswell as the agent selected. Generally, about 0.01 to about 10 weightpercent, preferably about 0.01 to about 5.0, more preferably about 0.01to about 1 weight percent of the coupling agent is used based on theweight of the coating solution. By coating solution is meant thesynthetic antimicrobial peptide with one or more of the solvent,coupling agent, buffer, and the like. Typically, the amount of coatingsolution used per lens will be about 1 ml to about 10 ml, preferablyabout 1 ml to about 5 ml, more preferably about 1 ml to about 2 ml perlens.

In the processes of the invention, a coating effective amount ofsynthetic antimicrobial peptide, such as L-melimine, protattin, orcombinations thereof is used meaning an amount that when contacted withthe surface is sufficient to coat the surface so as to impart thedesired antimicrobial properties to the surface. By antimicrobialproperties is meant either or both the ability to significantly reduce,meaning by greater than about 50 percent, either or both the amount ofbacteria adhering to the surface and the growth of bacteria adhered tothe surface. In the case of contact lenses, generally, the amountcontacted with the lens is about 1 μg to about 10 mg, preferably about10 μg to about 1 mg per lens. The amount of coating resulting percontact lens is about 50 to about 1000 μg. In cases in whichcombinations of L-melimine is used, the amount of L-melimine usedpreferably is between about 250 μg/lens to about 1000 μg/lens.

Temperature and pressure are not critical to the processes of theinvention and the process may be conveniently carried out at roomtemperature and pressure. The contact time used will be a length of timesufficient to coat the surface to the extent desired. Preferably,contact time is about 60 seconds to about 24 hours.

Following contacting, the surface may be washed with water or bufferedsaline solution to remove unreacted synthetic antimicrobial peptide andsolvent. One ordinarily skilled in the art will recognize that thepolymer for producing the surface to be coated by the method of theinvention may contain other monomers and additives. For example,ultra-violet absorbing monomers, reactive tints, processing aids, andthe like may be used.

The invention will be further clarified by a consideration of thefollowing, non-limiting examples.

Example 1

To assess the effect of cationic proteins/peptides in solution againstbacterial cells, Psudomonas aeruginosa 6294 and Staphylacoccus aureus 31cells were grown as for 18 hours in Tryptone Soya Broth (TSB) at 35° C.The cells were then harvested by centrifugation and washed twice inphosphate buffered saline (PBS; NaCl 8 g/l; KCl 0.2 g/l; Na₂HPO₄ 1.15g/l; KH₂PO₄ 0.2 g/l). The cells were then re-suspended to OD 0.1 at 660nm in PBS. The cationic peptide was serially diluted from 1 mg/ml anddilutions were added to wells in a 96 well microtitre plate or into 5 mldisposable test-tubes. Controls were bacteria without peptides. Bacteriawere incubated for 18 hours at 35° C. and turbidity was measured at 660nm. A lack of turbidity in the test-tube corresponding to the peptidedilution was considered to be the minimum inhibitory concentration(“MIC”).

TABLE 1 S.a. S.a. P.a P.a Peptide Sequence 31 CK5 6284 15442 MelimineT L I S W I K N K R K Q R P  125  250 R R R V S R R R R R R G G R R R RProttatin R P R V S R R R R R R G G 1000  500 500 1000R R R R T L I S W I K N K R K Q Mellitin T L I S W I K N K R K Q R R R R(15-26) Protamine R P R V S R R R R R R G G R R R R R 1000 R R (1-17)Protamine PRRRRSSSRPVRRRRRPRV R ND R ND SRRRRRRGGRRRR MellitinGIGAILKVLATGLPTLISWIKNKRKQ   15 ND R ND Values expressed as the minimumconcentration in μg/ml required to inhibit bacterial growth R =resistant, highest concentration of peptide = 1000 ug/Ml; ND = NOTdetermined S.a. 31 is S. aureus 31 S.a. CK5 is S. aureus CK5 P.a 6294 isP. aeruginosa 6294 P.a 15442 is P. aeruginosa 15442

The data in Table 1 indicate that when measured using the conventionaldoes setting method of the solution tube method, Melimine in solutionhas an MIC for S. aureus of between 60 and 250 μg/ml but that P.aeruginosa is resistant to this peptide up to 1000 μg/ml (the highestconcentration used).

Example 2

For conducting viable counts, Etafilcon A lenses were removed from themanufacturers vials, washed three times in 1 ml PBS, dried and thencoated by pipetting 500 μg L-Melimine onto the contact lenses (meliminewas dissolved in 20 distilled water and air dried onto contact lenses)overnight at ambient temperature in a fume hood. The number of lensesused for each strain is shown in Table 2 in parenthesis. Lenses wererehydrated in PBS for 10 minutes and 0.5 ml of 1×10⁸ bacterial cells/ml,including gram-positive S. aureus and gram-negative P. aeruginosa wereadded to the lenses. After incubation at ambient temperature for either10 min or 5 hours, the lenses were washed three times in PBS to removenon-adherent or loosely adherent bacteria. Lenses were then homogenizedusing tissue homogenizer and 1 ml PBS until lens disintegration (about1-2 minutes). Serial dilutions were then made according to the techniqueof Miles and Misra and aliquots (20 μL) plated out on nutrient agar.After incubation overnight at 37° C., viable bacteria were determinedand results expressed as colony forming units/mm² after calculation ofthe surface area of the lens (approximately 310 mm²). The effects arecalculated based upon comparison to adhesion control lens that was notcoated with cationic protein/peptide before bacterial adhesion testing.The results are shown on Table 2.

TABLE 2 Bacterial Strain % reduction at 10 min % reduction at 5 hrs P.aeruginosa 6294 44 ± 30 (4) 95 ± 2 (3) P. aeruginosa 15442 31 ± 38 (5)78 ± 16 (3) S. aureus 31 62 ± 32 (5) 77 ± 21 (3) S. aureus CK5 86 ± 18(4) 86 ± 7 (4)

After both 10 minutes and five hours exposure, L-Melimine was able tosignificantly reduce the number of bacteria on the hydrogel polymer. Thedata show the lenses coated with 500 μg of L-Melimine reduced the numberof microorganisms adhered to a hydrogel polymer by at least about 80%for the bacteria strains tested.

Example 3

Etafilcon lenses, commercially available from Johnson & Johnson asACUVUE® Brand contact lenses) were soaked as in Example 2, above, exceptthat the L-melimine concentration was varied: 125 μg, 250 μg or 500μg/lens. After absorption of L-Melimine onto the hydrogel polymer, thelenses were rehydrated with PBS, and exposed to S. aureus or P.saeruginosa for either 10 minutes or five hours, as described in Example2. After exposure, the polymer was evaluated for numbers ofmicroorganisms the standard Miles and Misra plate count assay.

TABLE 3 Bacterial % reduction at 10 min % reduction at 5 hrs Strain 125μg 250 μg 500 μg 125 μg 250 μg 500 μg P. aeruginosa −19 ± 41 27 ± 15 44± 30 60 ± 40 77 ± 11 93 ± 2  6294 (4) (4) (4) (8) (4) (3) S. aureus 31−1361 ± 1745 −127 ± 254  62 ± 32 −81 ± 98  −163 ± 266  77 ± 21 (4) (3)(5) (5) (3) (3) Number of lenses tested shown in ( )

The results in Table 3 show that, with a decreased concentration ofL-Melimine, there was a corresponding decrease in the numbers ofmicroorganisms that were inhibited.

Example 4

Stock solutions (50 mg/ml) were prepared containing the L-Protattin indistilled water. 500 μg of the stock solution was adsorbed to a contactlens made from Etafilcon A as for L-melamine in Example 2 for 18 hoursat ambient temperature in a fume hood. The subsequent experimentaldetails are as described in Example 2, above and viable bacteria wasmeasured. Results are shown in Table 4, below.

TABLE 4 Bacterial Strain % reduction at 10 min % reduction at 5 hrs P.aeruginosa 6294 97 ± 5 (6) 57 ± 39 (6) P. aeruginosa 15442 94 ± 4 (8) 63± 32 (7) S. aureus 31 64 ± 18 (6) 43 ± 33 (6) S. aureus CK5  6 ± 62 (7) 3 ± 25 (7) Number of lenses tested shown in ( )

The data in Table 4 shows a reduction in microorganisms attached to ahydrogel polymer with the Protattin peptide. However, in comparison withthe L-Melimine data in Example 2, the L-Melimine is a preferred order ofthe amino acid sequences as it was more effective than Protattin atreducing bacterial adhesion and colonisation to contact lenses,particularly after several hours.

Example 5

A heat stability study was conducted to evaluate whether post-exposureof the L-Melimine to autoclaving would alter the ability of the peptideto prevent bacterial growth.

Bacteria (P. aeruginosa 6294 and S. aureus 31) were grown for 18 hoursat 35° C. in TSB, then washed twice in PBS. The bacteria wereresuspended in PBS to an optical density of 0.1 at 660 nm. L-Meliminewas dissolved in PBS to a concentration of 1000 μg/ml and autoclaved at121° C. for 15 minutes. Equal volumes of bacterial suspension andL-Melimine were then added together and incubated at 35° C. for periodsof up to 72 hours. Controls were non-autoclaved L-Melimine. After 24, 48and 72 hours, aliquots of the bacteria/L-Melimine solutions were removedand the number of viable bacteria were counted using the standard Milesand Misra plate count assay. Tables 5A and 5B show the results.

TABLE 5A Heat stability of Melimine in solution against P. aeruginosa6294 % inhibition of growth Treatment @ 24 hours @ 48 hours @ 72 hoursNone 100 (5) 100 (3) 100 (2) Autoclaved 100 (3) 100 (3) 100 (2) Numberof lenses tested shown in ( )

TABLE 5B Heat stability of Melimine in solution against S. aureus 31 %inhibition of growth Treatment @ 24 hours @ 48 hours @ 72 hours None 100(5) 100 (3) 99 (2) Autoclaved 100 (3) 100 (3) 99 (2) Number of lensestested shown in ( )All standard deviations were 1 or less. Autoclave treatment of theL-Melimine did not result in any reduction in activity.

Example 6

Bacteria (Streptococcus pneumoniae and Serratia marcescens) were grownas described in Example 1. Contact lenses were prepared as is Example 2and allowed to soak in the bacteria solution for 10 minutes. Afteradhesion, the lenses were processed as described in Example 2 and the %reduction in bacteria as compared to a control lens which was notL-melimine treated was measured. The results are shown in Table 6,below.

TABLE 6 Bacterial Strain % reduction at 10 min S. pneumoniae 68 ± 7 (3)S. marcescens 43 ± 36 (4) Number of lenses tested shown in ( )

The data in Table 6 shows that L-Melimine was able to reduce theadhesion of S. pneumoniae and S. marcescens to contact lenses. Thesebacteria have been isolated from contact lenses at the time of anadverse event.

Example 7

Three contact lenses (Etafilcon A) were washed twice in 0.1 M sodiumacetate buffer pH 5.0 then resuspended in 2 ml 0.1M sodium acetatebuffer pH 5.0 with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (“EDC”) at a final concentration of 2 mg/ml in sodiumacetate buffer pH 5.0 and allowed to react for 15 minutes at roomtemperature. Contact lenses were then washed three times in PBS pH 7.4.Contact lenses were then resuspended in 1 mg/ml L-Melimine in PBS andincubated for two hours at 37° C. with mixing. The contact lenses werewashed four times in PBS and the bound peptide quantified by a methoddeveloped by Cole and Ralston (1994). Contact lenses were stained for2-24 hours using filtered 0.025% Coomassie stain in 10% acetic acid and10% iso-propanol at 37° C. Lenses were destained in 10% acetic acid and10% iso-propanol at 37° C. Lenses were then extracted in 25% Pyridineovernight. The extracted solutions were analysed in thespectrophotometer at A600 using 25% Pyridine as a blank. L-Meliminequantification was determined by correlating extracts against a standardcurve constructed by pipetting known amounts of L-Melimine on semi-driedacrylamide gels and extracting as above, this method extracts allamounts of peptide from the gel. Approximately 18 μg/lens of Meliminewas bound on to contact lenses using this method.

The lenses were exposed to bacteria (P. aeruginosa for 10 minutes at 35°C.) and analyzed as described in Example 2. The lenses showed a 66±3%reduction in bacterial adhesion.

Example 8

Contact lenses were coated and dried (as in Example 2) and D-Melimineapplied at a concentration of either 250 μg per lens or 500 μg per lens.D-melimine was made using D-amino acids as for L-melimine using standardpeptide synthesis techniques (e.g. Fmoc). Assays for measuringinhibition of bacterial adhesion were performed as in Example 2.

TABLE 7 % Reduction L-melimine % reduction @ 250 μg/ % reduction @ 500μg/ Bacterial lens lens Strain 10 min 5 hrs 10 min 5 hrs P. aeruginosa 27 + 15  77 + 11 (4) 44 + 30 93 + 2 (3) 6234 (4) (4) S. aureus 31 127 +254 163 + 266 62 + 32 77 + 21 (3) (3) (3) (5)

TABLE 8 % reduction D-melimine % reduction @ 250 μg/ % reduction @ 500μg/ Bacterial lens lens Strain 10 min 5 hrs 10 min 5 hrs P. aeruginosa 22 + 49  82 + 16  66 + 36  81 + 11 6234 (4) (5) (2) (2) S. aureus 31641 + 548 621 + 433 495 + 677 1761 + 2599 (4 (5) (2) (2)

At ten minutes and 250 μg/lens both L- and D-Melimines had littlestatistically measurable activity towards S. aureus (Tables 7 and 8).Additionally, there was no difference in activity against P. aeruginosafor the D-enantiomer at 250 μg/lens in comparison with the L-Melimine.At a concentration of 500 μg/lens, the L-entiomer showed activityagainst both P. aeruginosa and S. aureus, while D-enantiomer showedstatistically consistent activity towards P. aueruginosa.

Example 9

For in vitro cytotoxic testing, solutions of L-Melimine were prepared inPBS (500 μg/ml, 250 μg/ml, 125 μg/ml and 62.5 μg/ml). Solutions werethen diluted 1:3 in tissue culture media (EMEM) and added to murine L929cells for 48 hours. Cells are rinsed in saline then harvested using0.25% trypsin for 3-5 minutes at ambient temperature. 1 ml of saline isthen added to cells and trypsin mixture, a 500 μl aliquot is added to19.5 ml of saline and counted in a coulter counter in triplicate.Results are compared to unperturbed cultures as well as positive(cyotoxic) controls. Results are expressed as percentage of non-viablecells (100-number of cells in sample/number of cells in unperturbedcells×100). In addition, the ability of the peptides to lyze sheep redblood cells was examined. The sheep red blood cells were washed andresuspended in PBS. Peptides at various concentrations were added to thered blood cells and incubated at 37° C. for 4 h. 100% lysis was obtainedusing distilled water and 0% lysis was obtained using PBS. Lysis wasmeasured by measuring the amount of haemoglobin released into the PBSafter centrifugation to remove whole red blood cells.

TABLE 9 L-Melimine conc. (μg/ml) % cell inhibition 500 44 250 26 125 862.5 20

L-Melimine was found to be marginally cytotoxic at 500 μg/ml andnon-cytotoxic at all other concentrations (Table 9). As shown in FIG. 1,Melittin caused extensive hemolysis of red blood cells, whereas allother peptides/proteins caused much less hemolysis. L-Melimine causedthe least amount of hemolysis of any peptide/protein. These resultsindicate that Melimine may be used on contact lenses without causing anadverse reaction in eyes.

Example 10

L-Melimine was evaluated to determine its potential to induce resistancein P. aeruginosa and S. aureus. Prior to L-Melimine exposure, MICs ofboth strains were determined as in Example 1. The P. aeruginosa strainwas resistant at 1 mg/ml in Example 1, so starting concentration todetermine the MIC for L-Melimine when incubated with P. aeruginosa wasraised to 15 mg/ml and the MIC was determined to be 4 mg/ml, using theprocedure of Example 1. For the Example 10, about 25% of the MIC wasused as the subinhibitory concentration, which is shown in column 2 ofTable 10, below. Bacteria were grown overnight in TSB. Bacteria wereinoculated 1:100 into fresh TSB containing sub-inhibitory levels ofL-Melimine and incubated overnight. Bacteria from the broth containingsub-inhibitory levels (column 2, Table 9) were then re-incubated infresh TSB with the same concentration of L-Melimine. This was repeatedfor 30 consecutive days. After passaging, a MIC for the bacteria fromthe last passage was determined. Any increase in MIC would indicate apotential for bacteria to become resistant.

TABLE 10 Sub-inhibitory MIC Strain concentration MIC prior to passagepost-passage P. aeruginosa  1 mg/ml  4 mg/ml  4 mg/ml S. aureus 30 μg/ml125 μg/ml 63 μg/ml

The MIC P. aeruginosa did not change over time and shows no potentialfor resistance to Melimine. The MIC for S. aureus actually decreasedover the passage time, which indicates that in sub-inhibitory levelsthis strain is becoming more susceptible to this peptide. There is noindication for potential resistance to L-Melimine by either bacterium.Thus, the present invention provides cationic peptides withantibacterial activity and no indication for antibacterial resistanceover time. Thus, medical devices which are coated or treated with thesepeptides would be suitable for applications where repeated or long termuse and antibacterial activity are desired.

Example 11

L-Melimine was evaluated to determine its able to retain activity in thepresence of tears. L-Melimine was incubated in the presence of tears asfollows. Holes were punched in nutrient agar plates which were thenseeded with bacteria (P. aeruginosa 6294 or S. aureus 31). Tears pooledfrom normal human subjects (10 μl) were added into wells and allowed toabsorb into the agar for 15 minutes at ambient temperature. L-Meliminewas added at a concentration of 125 μg/well. The nutrient agar plateswere incubated overnight at 35° C., following incubation, the zone ofinhibition was measured and expressed in mm. The results are shown inTable 11, below.

TABLE 11 Zone of inhibition (mm) Strain Tears L-Melimine L-Melimine +Tears P. aeruginosa 0 6 ± 1 6.8 ± 1.8 S. aureus 0 7.25 ± 1.9  6.9 ± 0.9There was no significant increase or decrease in the activity ofL-Melimine when in the presence of tears suggesting that tears do notinterfere with the activity of this peptide.

Example 12

To a dry container housed in a dry box under nitrogen at ambienttemperature was added 30.0 g (0.277 mol) ofbis(dimethylamino)methylsilane, a solution of 13.75 ml of a 1M solutionof TBACB (386.0 g TBACB in 1000 ml dry THF), 61.39 g (0.578 mol) ofp-xylene, 154.28 g (1.541 mol) methyl methacrylate (1.4 equivalentsrelative to initiator), 1892.13 (9.352 mol) 2-(trimethylsiloxy)ethylmethacrylate (8.5 equivalents relative to initiator) and 4399.78 g(61.01 mol) of THF. To a dry, three-necked, round-bottomed flaskequipped with a thermocouple and condenser, all connected to a nitrogensource, was charged the above mixture prepared in the dry box.

The reaction mixture was cooled to 15° C. while stirring and purgingwith nitrogen. After the solution reaches 15° C., 191.75 g (1.100 mol)of 1-trimethylsiloxy-1-methoxy-2-methylpropene (1 equivalent) wasinjected into the reaction vessel. The reaction was allowed to exothermto approximately 62° C. and then 30 ml of a 0.40 M solution of 154.4 gTBACB in 11 ml of dry THF was metered in throughout the remainder of thereaction. After the temperature of reaction reached 30° C. and themetering began, a solution of 467.56 g (2.311 mol)2-(trimethylsiloxy)ethyl methacrylate (2.1 equivalents relative to theinitiator), 3636.6. g (3.463 mol) n-butylmonomethacryloxypropyl-polydimethylsiloxane (3.2 equivalents relative tothe initiator), 3673.84 g (8.689 mol) TRIS (7.9 equivalents relative tothe initiator) and 20.0 g bis(dimethylamino)methylsilane was added.

The mixture was allowed to exotherm to approximately 38-42° C. and thenallowed to cool to 30° C. At that time, a solution of 10.0 g (0.076 mol)bis(dimethylamino)methylsilane, 154.26 g (1.541 mol) methyl methacrylate(1.4 equivalents relative to the initiator) and 1892.13 g (9.352 mol)2-trimethylsiloxy)ethyl methacrylate (8.5 equivalents relative to theinitiator) was added and the mixture again allowed to exotherm toapproximately 40° C. The reaction temperature dropped to approximately30° C. and 2 gallons of THF were added to decrease the viscosity. Asolution of 439.69 g water, 740.6 g methanol and 8.8 g (0.068 mol)dichloroacetic acid was added and the mixture refluxed for 4.5 hours tode-block the protecting groups on the HEMA. Volatiles were then removedand toluene added to aid in removal of the water until a vaportemperature of 110° C. was reached.

The reaction flask was maintained at approximately 110° C. and asolution of 443 g (2.201 mol) TMI and 5.7 g (0.010 mol) dibutyltindilaurate were added. The mixture was reacted until the isocyanate peakwas gone by IR. The toluene was evaporated under reduced pressure toyield an off-white, anhydrous, waxy reactive monomer. The macromer wasplaced into acetone at a weight basis of approximately 2:1 acetone tomacromer. After 24 hrs, water was added to precipitate out the macromerand the macromer was filtered and dried using a vacuum oven between 45and 60° C. for 20-30 hrs.

Example 13

A reaction mixture was formed by adding 100 parts of the componentsshown in Table 12, in the amounts shown in Table 12 with 20 parts3,7-dimethyl-3-octanol. Specifically, in the following order macromer,Norbloc 7966, diluent, TEGDMA, HEMA, DMA, TRIS, and mPDMS were added toan amber flask. These components were mixed at 170-300 rpm, at 50-55°C., for 90 to 180 minutes. While maintaining mixing, blue HEMA was addedand the components mixed for a further 20 to 75 minutes (at 170-300 rpm,50-55° C.). Still with mixing, PVP was added and the mixture stirred foranother 20 to 140 minutes (at 170-300 rpm, 50-55° C.). Lastly, withcontinual mixing, CGI 1850 (Irgacure 1850) was added.

TABLE 12 Component Weight Percent Macromer (Ex 12) 18.95 TRIS 14.7 DMA27.4 MPDMS 29.5 NORBLOC 2.1 CGI 1850 1.1 TEGDMA 1.1 HEMA 5.3

Pentafluorophenyl methacrylate (OPfp) (0.5 wt. %) was added to thereaction mixture. The reaction mixture was mixed vigorously forapproximately 10 minutes (or until the solution appeared clear andevenly mixed) and the then degassed, on high vacuum, until no airbubbles were visible in the reaction mixture (about 20 minutes). Thereaction mixtures were placed into thermoplastic contact lens molds, andirradiated using Philips TL 20W/03T fluorescent bulbs at 50° C., forabout 50 minutes in an N₂ atmosphere. The lenses were demolded inDowanol® DPMA (DPMA, commercially available from Aldrich). Lenses werewashed up to five times with DPMA. Each wash lasted about 120 minutes.The lenses were individually placed into vials containing 2 mL of asolution with 2.5 mg/mL melimine and 0.05 weight percent ofN,N-diisopropylethylamine (DIPEA) in N,N-dimethylformamide (DMF). Thevials (containing the lenses and solution) were stoppered with graybutyl stoppers and then incubated in an Incubator/Shaker for 18 hours at37° C., shaking continuously at 100 rpm.

After incubation, the solvent from each of the vials was removed.Approximately 9 mL of fresh DMF solvent was then added to each vial.After 1 hour, the solvent was removed and fresh DMF solvent was re-addedat the same volume. This process was repeated a total of 4 times each at1 hour intervals. After the fourth solvent change out, the lenses wereplaced directly into DI water at room temperature and washed 4 times at1 hour intervals. After the fourth wash, the lenses were placed intopacking solution at room temperature for 1 hour and then autoclaved for30 minutes at 121° C. Lens properties were measured and are shown inTable 13, below.

Example 14

Example 13 was repeated except that coupling additives were added to themelimine-containing coating solution and that the concentration of themelimine-coating solution was changed. So, exactly as per Example 13,the lenses after being released and washed in DPMA solvent, were placedinto individually into vials containing 3 mL of a solution of 5 mg/mL ofN-hydroxybenzotriazole (HOBt) in DMF. Using a calibrated pipettor, 50 μLof diispropylcarbodiimide (DIC) was added to each vial. After 20 to 60minutes at room temperature, 1 mL of a 3 mg/mL melimine in DMF solutioncontaining 0.05 weight percent of N,N-diisopropylethylamine (DIPEA) wasadded to each vial using a calibrated Eppendorf pipettor. The vials werestoppered with gray butyl stoppers. The lenses were then incubated in anIncubator/Shaker for about 19 hours at 37° C. with shaking continuouslyat 100 rpm.

After incubation, the solvent from each of the vials was removed.Approximately 9 mL of fresh DMF solvent was then added to each vial.After 1 hour, the solvent was removed and fresh DMF solvent was re-addedat the same volume. This process was repeated a total of 4 times each at1 hour intervals. After the fourth solvent change out, the lenses wereplaced directly into DI water at room temperature and washed 4 times at1 hour intervals. After the fourth wash, the lenses were placed intopacking solution at room temperature for 1 hour and then autoclaved for30 minutes at 121° C. Lens properties were measured and are shown inTable 13, below. Standard deviations are shown in parenthesis.

TABLE 13 Property Control 1 Ex 13 Ex 14 Water Content (%) 35.1(0.2) 35.6(0.3)  35.4(0.2) Modulus (psi) 113.0(11.9)  119.9(10.3)  132.9(13.0)Elongation (%) 176.5(75.1)  128.2(67.8)  213.1(56.6) Tensile Strength(psi) 93.1(45.8) 72.4(43.6) 127.2(35.5) Toughness (psi) 91.7(60.0)53.5(59.0) 131.0(62.2) DCA (°) 96(20) 95(13)  67(25) Melimine N/A ≈120≈140 Concentration (ug/lens)

Example 15

Lenses made in Examples 13 and 14 were removed from vials and washedthree times in 5 mL of PBS for 5 minutes each wash. Uncoated and lenseshaving the same substrate formulation were used as a control. Fourlenses were exposed to bacteria (P. aeruginosa or S. aureus) at 35° C.for the times shown in Table 14, below and analyzed as described inExample 2.

TABLE 14 Example 13 Example 14 Mean SD Mean SD P. aeruginosa (% redxn)Time 10 min 50 0 45 16  5 hrs 58 1.6 46 17 S. aureus (% redxn) Time 10min 34 18 47 31  5 hrs 32 11 33 27

Examples 16-19

Example 14 was repeated except that the concentrations of themelimine-coating solution and the wash procedure were changed. So,exactly as per Example 14, the lenses after being released and washed inDPMA solvent, were placed into individually into vials containing 3 mLof a solution of 5 mg/mL of N-hydroxybenzotriazole (HOBt) in DMF. Usinga calibrated pipettor, 50 μL of diispropylcarbodiimide (DIC) was addedto each vial. After about 60 minutes at room temperature, 1 mL of amelimine-coating solution of various concentration listed in Table 15,in DMF containing 0.05 weight percent of N,N-diisopropylethylamine(DIPEA) was added to each vial using a calibrated Eppendorf pipettor.The vials were stoppered with gray butyl stoppers. The lenses were thenincubated in an Incubator/Shaker for about 19 hours at 37° C. withshaking continuously at 100 rpm.

After incubation, lenses were transferred to a 400 mL beaker containing300 mL of fresh DMF and a magnetic stirrer. The lenses were stirred inthe DMF for 1 hour. This process was repeated three more times (fourtimes total). After the fourth solvent change out, the 300 mL of DIwater was added to the beaker and the lenses washed a total of fourtimes with DI water. After the fourth wash, the lenses were placed intovials containing packing solution and then autoclaved for 30 minutes at121° C. Lens properties were measured and are shown in Table 15, below.Standard deviations are shown in parenthesis.

TABLE 15 Property Control 2 Ex 16 Ex 17 Ex 18 Ex 19 Coating Solution 00.75 1.50 3.00 6.00 Conc. (mg/mL) Water 35.8(0.3)  N/M 35.5(0.3) 35.6(0.3)  N/M Content(%) Modulus(psi) 95(8)  N/M 111(13)  107(8) 108(12)  Elongation(%) 134(70)  N/M 164(82)  161(66)  155(55)  Tensile55(28) N/M 80(43) 77(34) 72(25) Strength(psi) Toughness(psi) 44(42) N/M77(67) 68(53) 59(39) DCA (°) N/M N/M N/M N/M N/M Melimine 63 128 219 334Conc. (ug/lens) N/M indicates not measured.

Example 20

Lenses made in Examples 16 to 19 were removed from vials and washedthree times in 5 mL of PBS for 5 minutes each wash. Uncoated and lenseshaving the same substrate formulation were used as a control. Fourlenses were exposed to bacteria (P. aeruginosa or S. aureus) at 35° C.for the times shown in Table 16, below and analyzed as described inExample 2. Standard deviations are shown in parenthesis

TABLE 16 Ex 16 Ex 17 Ex 18 Ex 19 P. aeruginosa (% redxn) Time 10 min.  5(33)  2 (22) −31 (35)  5 (19)  5 hrs 37 (12) 27 (17)  20 (10)  27 (33)S. aureus (% redxn) Time 10 min. −34 (70)   3 (72) −17 (57) −55 (66)  5hrs −5 (32) −9 (59) −17 (38) −55 (3) 

Example 21

To a 100 mL round bottom flask equipped with a magnetic stirrer andcontaining 0.60 g melimine, was added 50 mL of N,N-dimethylformamide(DMF). The homogeneous solution was stirred, under nitrogen, at 25° C.,for 30 minutes. N,N-Diisopropylethylamine (DIPEA, 33 μL) was added tothe solution. The solution was placed in an ice bath and stirred, stillunder nitrogen. After 20 minutes, methacryloyl chloride (19 μL) wasadded to the solution. The solution was allowed to come to roomtemperature and monitored by mass spectrometry. After about 20 hours, 19μL more of methacryloyl chloride was added to the solution and thesolution stirred, at room tempertaure. After about another 20 hours, DMFwas evaporated from the flask and the residue dissolved in DI water anddialyzed against 2×4 L of DI-water using dialysis tubing (benzoylated,cellulose dialysis tubing, molecular weight cut off 2,000, availableform Sigma). The dialyzed portion was freeze dried to give about 250 mgof methacryloylated melimine as a fluffy white powder.

Example 22

Methacryloylated melimine (Example 21, 74.9 mg) was added to 3.12 gramsof DMA and the mixture sonicated at 40° C. until all of the melimineappeared to dissolve (about 1 hour). This melimine/DMA solution was usedto make a reaction mixture. A reaction mixture was formed by adding 100parts of the components shown in Table 17, in the amounts shown in Table17 with 20 parts 3,7-dimethyl-3-octanol. Specifically, in the followingorder macromer, Norbloc 7966, diluent, TEGDMA, HEMA, melimine/DMAsolution, TRIS, and mPDMS were added to an amber flask. These componentswere mixed at 170-300 rpm, at 50-55° C., for 90 to 180 minutes. Whilemaintaining mixing, blue HEMA was added and the components mixed for afurther 20 to 75 minutes (at 170-300 rpm, 50-55° C.). Still with mixing,PVP was added and the mixture stirred for another 20 to 140 minutes (at170-300 rpm, 50-55° C.). Lastly, with continual mixing, CGI 1850(Irgacure 1850) was added.

TABLE 17 Component Weight Percent Macromer (Ex 12) 18.95 TRIS 14.7Melimine/DMA solution 27.4 MPDMS 29.5 NORBLOC 2.1 CGI 1850 1.1 TEGDMA1.1 HEMA 5.3

The reaction mixture was degassed, on high vacuum, until no air bubbleswere visible in the reaction mixture (about 20 minutes). The reactionmixtures were placed into thermoplastic contact lens molds, andirradiated using Philips TL 20W/03T fluorescent bulbs at 50° C., forabout 60 minutes in an N₂ atmosphere. After irradiation, the molds wereopened and the lenses released in 60% isopropanol/water and then leachedin IPA/DI to remove any residual monomers and diluent. Lenses weretransferred to vials containing packing solution and then autoclaved for30 minutes at 121° C. Lens properties were measured and are shown inTable 18.

TABLE 18 Property Control 3 Ex 22 Water Content (%) 37.7(0.3) 38.7(0.3)Modulus (psi) 87(9) 87(6) Elongation (%) 258(46) 279(56) TensileStrength (psi) 108(27) 132(42) Toughness (psi) 131(46) 166(63) MelimineConcentration ≈3 ≈70 (ug/lens)

Example 23

Lenses made in Example 22 were removed from vials and washed three timesin 5 mL of PBS for 5 minutes each wash. Lenses having the same substrateformulation, but without the methacryloylated melimine were used as acontrol. Four lenses were exposed to bacteria (P. aeruginosa or S.aureus) at 35° C. for the times shown in Table 19, below and analyzed asdescribed in Example 2.

TABLE 19 Mean SD P. aeruginosa (% redxn) Time 10 min. −13 14  5 hrs 5 36S. aureus (% redxn) Time 10 min. −20 87  5 hrs −31 25

Example 24

To a 500 mL round bottom flask equipped with a magnetic stirrer andcontaining 3.00 g melimine, was added 120 mL of N,N-dimethylformamide(DMF). The homogeneous solution was stirred, under nitrogen, at 25° C.,for about 5 minutes. N,N-Diisopropylethylamine (DIPEA, 170 μL) was addedto the solution. The solution was placed in an ice bath and stirred,still under nitrogen. After 30 minutes, methacryloyl chloride (95 μL)was added to the solution. The solution was allowed to come to roomtemperature. After about 20 hours, the reaction was complete, asmonitored by mass spectrometry. DMF was evaporated from the flask andthe residue dissolved in about 10 mLs DI water and dialyzed against 3×4L of DI-water using dialysis tubing (benzoylated, cellulose dialysistubing, molecular weight cut off 2,000, available form Sigma). Thedialyzed portion was freeze dried to give about 940 mg ofmethacryloylated melimine as a fluffy white powder.

Examples 25 to 27

Example 22 was repeated using the methacyloylated melamine of Example 24in the amounts shown in Table 20, below. Lens properties were measuredand are shown in Table 20. Standard deviations are shown in parenthesis

TABLE 20 Property Control 4 Ex 25 Ex 26 Ex 27 Methacryloylated 0 56.26112.5 225.2 Melimine added to 15 g of reaction mixture (mg) WaterContent (%) 36.2(0.3) 34.2(0.3)  33.8(0.3) 33.1(0.3) Modulus (psi) 80(17) 101(12)  103(13) 111(13) Elongation (%) 210(99) 189(74)  174(69)205(49) Tensile Strength (psi)  71(37) 77(35)  68(25)  82(18) Toughness(psi)  89(67) 84.(60)   67(50)  90(37) Contact Angle (adv. in 114(13)73(25) 53(5)  69(13) packing)

Example 28

Lenses made in Examples 25 to 27 were removed from vials and washedthree times in 5 mL of PBS for 5 minutes each wash. Lenses having thesame substrate formulation, but without the methacryloylated meliminewere used as a control. Four lenses were exposed to bacteria (P.aeruginosa or S. aureus) at 35° C. for the times shown in Table 21,below and analyzed as described in Example 2. Standard deviations areshown in parenthesis.

TABLE 21 Ex 25 Ex 26 Ex 27 P. aeruginosa (% redxn) Time 10 min.  5 (15)12 (7)   −90 (113)  5 hrs 78 (1)  74 (2)  78 (1) S. aureus (% redxn)Time 10 min. −39 (10)   35 (27) −156 (214)  5 hrs −17 (101) 38 (62) 65(2)

1. A process for manufacturing a biomedical device comprising the stepof contacting at least one surface of said biomedical device with acoating effective amount of at least one synthetic antimicrobial peptidecomprising three segments, A, B and C, in any order, wherein segment Ais a peptide having the sequence SEQ ID NO. 1; segment B is a peptidehaving the sequence SEQ ID NO. 2; and segment C is a linking group of upto 10 amino acids, and which does not inhibit the antimicrobial activityof the peptide or induce toxicity in mammalian cells.
 2. The process ofclaim 1 wherein segment C has a formula —HN—(CR¹R²)_(n)—CO— wherein n isan integer between 1 and 21, R¹ and R² are independently selected fromthe group consisting of H, straight or branched alkyl groups having 1 to4 carbon atoms, straight or branched hydroxy groups having 1-2 carbonatoms, straight or branched alkylthio groups having 1 to 3 carbon atoms,carbamoyl groups having 1 to 3 carbon atoms, carboxy groups having 1 to3 carbon atoms, primary and secondary amino groups having 1 to 4 carbonatoms and 1 to 3 nitrogen atoms, benzyl, phenol, phenyl indoles andN,N-pyrroles.
 3. The process of claim 1 wherein n is an integer between1 and 10 and at least one of R¹ and R² is H.
 4. The process of claim 1wherein the A and B segments are in terminal positions and segment Ccomprises up to 5 amino acids.
 5. The process of claim 1, wherein the atleast one synthetic antimicrobial peptide comprises melimine, protattinand mixtures thereof.
 6. The process of claim 1 wherein the biomedicaldevice is a contact lens.
 7. The process of claim 6, further comprisingthe step of contacting the at least one surface with a couplingeffective amount of a coupling agent.
 8. The process of claim 5, whereinsaid synthetic antimicrobial peptide comprises L-melimine.
 9. Theprocess of claim 1 further comprising the step of bonding said syntheticantimicrobial peptide to said device.
 10. The process of claim 9 whereinsaid bonding step is conducted via direct reaction of reactive groups insaid device with reactive groups in said synthetic antimicrobial peptideor reaction of said device and synthetic antimicrobial peptide with acoupling agent.
 11. The process of claim 10 wherein said bonding step isconducted via reaction with at least one coupling agent and said atleast one coupling agent is selected from the group consisting ofN,N′-carbonyldiimidazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,dicyclohexyl carbodiimide,1-cylcohexyl-3-(2-morpholinoethyl)carbodiimide, diisopropyl carbodiimideand mixtures thereof and N-hydroxysuccinimide esters, imidoesters,difluorobenzene derivatives, bromofunctional aldehydes, bis epoxides andmixtures thereof.
 12. The process of claim 9 further comprising the stepof incorporating at least one latent reactive component into a reactivemixture from which said device is formed.